We have cloned two novel adipocyte-specific enzymes that catalyze opposing reactions in triacylglycerol (TAG) metabolism. We identified these enzymes by employing the unique criteria of adipocyte-specific expression patterns and the presence of structural motifs for lipid metabolism, and we have named them desnutrin and adipocyte phospholipid acyltransferase (APAT). Desnutrin is an adipocyte-specific TAG lipase whereas APAT is an adipocyte-specific acyltransferase. Expression of these two novel enzymes in adipocytes is inversely regulated during fasting/feeding, a prediction of their opposing physiological roles in adipocyte TAG metabolism. Together, these enzymes may govern TAG storage and, therefore, adipocyte size. Desnutrin has been characterized as a TAG lipase increasing TAG hydrolysis in cultured cells but its function in vivo needs to be demonstrated. In contrast, the importance of APAT in TAG storage in adipocytes has been shown by in vivo studies with knockout mice, but in vitro characteristics of APAT have not been established. Aim 1 is to characterize desnutrin and APAT in vitro for their substrate specificity as well as their preference for fatty acids in terms of chain length and saturation using purified enzyme preparations. In addition, physiological functions of these enzymes will be examined in cultured cells overexpressing desnutrin and APAT or in cells deficient in these enzymes. Aim 2 is to examine their subcellular localization by using antibodies that we raised, by expressing desnutrin or APAT as GFP- fusion proteins, and by classic subcellular fractionation. We will examine the potential translocation of desnutrin during lipolytic stimulation and the localization of APAT when esterification is favored. Possible involvement of phosphorylation/dephosphorylation mechanisms as well as interactions of desnutrin and APAT with other cellular proteins for translocation/regulation will be examined also. Aim 3 is to generate desnutrin and APAT null mice to demonstrate the functional significance of these enzymes and their effects on TAG metabolism/storage in adipose tissue and on glucose/insulin homeostasis in vivo. Elucidating the properties and functions of these two novel adipocyte-specific enzymes would be critical in understanding adipocyte TAG metabolism, and in developing future'therapeutic strategies against metabolic diseases such as obesity/diabetes.